The lipid landscape of cellular membranes changes with the development of disease such as infantile congenital heart failure, hypertension, and Alzheimer's. Also associated with these diseases are ionic currents passing through ion channels that regulate excitability in these cells. The intimate interaction of ion channels with te cell membrane is a major determinant of function, yet is not well characterized. Inwardly rectifying potassium (IKir) currents, found in cardiac myocytes and many other excitable cells play a vital role in setting the membrane potential and modulating membrane excitability. Until recently, analyses of lipid regulation of mammalian Kir2.1 and Kir2.2 ion channels were largely qualitative since these studies could only be performed in cells, where lipid composition is complex, difficult to control, and poorly understood. Biochemical properties of human Kir channels inferred from analysis performed on their bacterial homologues are limited since they share only ~30% sequence identity and are regulated differently by some membrane lipids. Thus, until recently, biochemical and structural analyses on eukaryotic Kir channels remained elusive. However, the PI has recently made a critical breakthrough in being able to express and purify functional human Kir channels in yeast. This breakthrough opens up several important avenues to understand regulation of Kir channels, as proposed in Aim 1 of this proposal. The expertise developed will then be used to assess the role of lipids in modulating representative members of two other K+ permeating channel sub-families (Kv1.2-2.1 and HCN channels) with topological and functional characteristics distinct from the Kir channel family (Aim 2). These particular sub-families have been selected for examination because of their important physiological role and, because despite some evidence that they may also be regulated by membrane lipids, little is known about lipid modulation of these channels. Thus, using a unique combination of biochemical techniques, 86Rb+ flux assays, electrophysiology, and molecular dynamics simulations, these two specific aims will address the unifying question: What are the molecular determinants and mechanisms by which lipids regulate K+ channels?